The evolutionarily conserved AHR has been studied historically for its role in environmental chemical- induced toxicity. With the demonstration of th AHR's role in several physiological functions comes the realization that even more organ systems may be impacted by AHR ligands than previously appreciated. Our computational analyses of primary human cells indicate Ahr upregulation during hematopoiesis and Ahr co- regulation with several genes critical to stem cells, erythroid cells, and megakaryocyte development. To further study the role of the AHR in human hematopoiesis, we developed a unique platform for the directed differentiation of pluripotent stem cells in chemically-defined, serum- and feeder cell-free culture conditions. This platform relies on the ability of non-toxic AHR agonists to efficiently produce virtually unlimited numbers of hemogenic endothelial cells (HECs), bi-potential hematopoietic progenitor cells (HPCs), hemoglobin- producing erythroid (Ery) cells, and polyploid megakaryocytes. Using this system, we generated compelling data supporting the central hypothesis that the AHR plays a critical role at several key decision points throughout normal hematopoietic cell development. As a corollary, we propose that environmental AHR ligands have the potential to alter this tightly regulated process. We propose three specific aims to test these hypotheses: Specific Aim 1: Map AHR-regulated fate decision points in human hematopoietic cell development. Using human iPSCs genetically engineered to express an AHR-driven reporter or inducible AHR-specific shRNA, we will construct a temporal map of AHR expression, activation, and function during nominal human hematopoietic cell differentiation, establishing a foundation for global analysis of the AHR- regulated transcriptome (Aim 2) and for validating a humanized mouse model (Aim 3). Specific Aim 2: Read genomic signatures of AHR-dependent cell fate decisions. This aim complements the functional analyses of Aim 1 by providing an unbiased, high resolution map of AHR-dependent transcriptional programs in HECs, HPCs and erythroblasts. Specific Aim 3: Determine the role of the AHR in human blood cell expansion and differentiation in an adoptive transfer animal model. Large numbers of luciferase-tagged and shAHR-expressing human iPSC-derived HPCs will be transplanted into immunocompromised mice and their differentiation and function fully characterized. In all three aims, the effects of putative endogenous and environmental AHR ligands on human erythroid- and megakaryocyte-lineage development will be compared. These studies are highly significant in that they use unique strategies to: 1) compare the effects of disparate AHR ligands on hematopoiesis, 2) reveal the basic biology behind AHR control of blood cell development, and 3) advance the technology towards the goal of generating clinical grade, transferable RBCs and platelets. They also exploit the combined expertise of the co-PIs in AHR signaling (Dr. Sherr) and stem cell biology (Dr. Murphy) and of Dr. Monti, a co-investigator, in computational biology.